Hematopoietic stem cells (HSCs) are generated from specialized endothelial cells of the embryonic aorta. Inflammatory factors are implicated in regulating mouse HSC development, but which cells in ...the aorta-gonad-mesonephros (AGM) microenvironment produce these factors is unknown. In the adult, macrophages play both pro- and anti-inflammatory roles. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation were involved in the AGM HSC-generative microenvironment. CyTOF analysis of CD45+ AGM cells revealed predominance of two hematopoietic cell types, mannose-receptor positive macrophages and mannose-receptor negative myeloid cells. We show here that macrophage appearance in the AGM was dependent on the chemokine receptor Cx3cr1. These macrophages expressed a pro-inflammatory signature, localized to the aorta, and dynamically interacted with nascent and emerging intra-aortic hematopoietic cells (IAHCs). Importantly, upon macrophage depletion, no adult-repopulating HSCs were detected, thus implicating a role for pro-inflammatory AGM-associated macrophages in regulating the development of HSCs.
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•Yolk-sac-derived macrophages are the most abundant hematopoietic cells in the AGM•Cx3cr1 mediates yolk-sac macrophage progenitor recruitment to the AGM niche•AGM macrophages dynamically interact with emerging intra-aortic hematopoietic cells•Pro-inflammatory AGM macrophages are positive regulators of HSC generation
HSC-independent macrophages derive from the early yolk-sac stages of embryonic hematogenesis. Mariani and colleagues demonstrate that specific pro-inflammatory embryonic HSC-independent macrophages recruited to the AGM (AGM-aMs) are crucial components of the AGM microenvironment, dynamically interact with emerging hematopoietic cells, and enhance HSC generation.
Whereas hundreds of cells in the mouse embryonic aorta transdifferentiate to hematopoietic cells, only very few establish hematopoietic stem cell (HSC) identity at a single time point. The Gata2 ...transcription factor is essential for HSC generation and function. In contrast to surface-marker-based cell isolation, Gata2-based enrichment provides a direct link to the internal HSC regulatory network. Here, we use iterations of index-sorting of Gata2-expressing intra-aortic hematopoietic cluster (IAHC) cells, single-cell transcriptomics, and functional analyses to connect HSC identity to specific gene expression. Gata2-expressing IAHC cells separate into 5 major transcriptomic clusters. Iterative analyses reveal refined CD31, cKit, and CD27 phenotypic parameters that associate specific molecular profiles in one cluster with distinct HSC and multipotent progenitor function. Thus, by iterations of single-cell approaches, we identify the transcriptome of the first functional HSCs as they emerge in the mouse embryo and localize them to aortic clusters containing 1–2 cells.
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•Single-cell iterations capture the transcriptome of the first functional HSCs in mouse•Gata2 connects the “inner” HSC regulatory network with single-cell function•Specific Gata2, cKit, and CD27 levels define all HSCs in embryonic aortic clusters•HSCs emerge from endothelium as single cells within aortic clusters of 1–2 cells
Vink and colleagues capture the transcriptome of the first functional HSCs in mouse by single-cell RNA-seq, index-sorting, and in vivo and invitro hematopoietic analyses. The HSC transcriptome is unique compared to HPCs, and heterogeneous expression of pivotal genes suggests that establishment of functional hematopoietic fate during cell emergence from embryonic aortic endothelium is stochastic.
BMI1 is a key component of the PRC1 (polycomb repressive complex-1) complex required for maintenance of normal and cancer stem cells. Its aberrant expression is detected in chronic myeloid leukemia ...and Ph+ acute lymphoblastic leukemia (ALL), but no data exist on BMI1 requirement in ALL cells. We show here that BMI1 expression is important for proliferation and survival of Ph+ ALL cells and for leukemogenesis of Ph+ cells in vivo. Levels of BIM, interferon-α (IFNα)-regulated genes and E2F7 were upregulated in BMI1-silenced cells, suggesting that repressing their expression is important for BMI1 biological effects. Consistent with this hypothesis, we found that: (i) downregulation of BIM or E2F7 abrogated apoptosis or rescued, in part, the reduced proliferation and colony formation of BMI1 silenced BV173 cells; (ii) BIM/E2F7 double silencing further enhanced colony formation and in vivo leukemogenesis of BMI1-silenced cells; (iii) overexpression of BIM and E2F7 mimicked the effect of BMI1 silencing in BV173 and SUP-B15 cells; and (iv) treatment with IFNα suppressed proliferation and colony formation of Ph+ ALL cells. These studies indicate that the growth-promoting effects of BMI1 in Ph+ ALL cells depend on suppression of multiple pathways and support the use of IFNα in the therapy of Ph+ ALL.
The transcription factor C/EBPα is more potent than C/EBPβ in inducing granulocitic differentiation and inhibiting BCR/ABL-expressing cells. We took a “domain swapping” approach to assess biological ...effects, modulation of gene expression, and binding to C/EBPα-regulated promoters by wild-type and chimeric C/EBPα/C/EBPβ proteins. Wild-type and N-C/EBPα+ C/EBPβ-DBD induced transcription of the granulocyte-colony stimulating factor receptor (G-CSFR) gene, promoted differentiation, and suppressed proliferation of K562 cells vigorously; instead, wild-type C/EBPβ and N-C/EBPβ+C/EBPα-DBD had modest effects, although they bound the G-CSFR promoter like wild-type C/EBPα and N-C/EBPα+C/EBPβ-DBD. Chimeric proteins consisting of the TAD of VP16 and the DBD of C/EBPα or C/EBPβ inhibited proliferation and induced differentiation of K562 cells as effectively as wild-type C/EBPα. Gene expression profiles induced by C/EBPα resembled those modulated by N-C/EBPα+C/EBPβ-DBD, whereas C/EBPβ induced a pattern similar to that of N-C/EBPβ+C/EBPα-DBD. C/EBPα activation induced changes in the expression of more cell cycle- and apoptosis-related genes than the other proteins and enhanced Imatinib-induced apoptosis of K562 cells. Expression of FOXO3a, a novel C/EBPα-regulated gene, was required for apoptosis but not for differentiation induction or proliferation inhibition of K562 cells.
The transcription factor C/EBPa is more potent than C/EBPb in inducing granulocitic differentiation and inhibiting BCR/ABL-expressing cells. We took a "domain swapping" approach to assess biological ...effects, modulation of gene expression, and binding to C/EBPa-regulated promoters by wild-type and chimeric C/EBPa/C/EBPb proteins. Wild-type and N-C/EBPa+ C/EBPb-DBD induced transcription of the granulocyte-colony stimulating factor receptor (G-CSFR) gene, promoted differentiation, and suppressed proliferation of K562 cells vigorously; instead, wild-type C/EBPb and N-C/EBPb+C/EBPa-DBD had modest effects, although they bound the G-CSFR promoter like wild-type C/EBPa and N-C/EBPa+C/EBPb-DBD. Chimeric proteins consisting of the TAD of VP16 and the DBD of C/EBPa or C/EBPb inhibited proliferation and induced differentiation of K562 cells as effectively as wild-type C/EBPa. Gene expression profiles induced by C/EBPa resembled those modulated by N-C/EBPa+C/EBPb-DBD, whereas C/EBPb induced a pattern similar to that of N-C/EBPb+C/EBPa-DBD. C/EBPa activation induced changes in the expression of more cell cycle- and apoptosis-related genes than the other proteins and enhanced Imatinib-induced apoptosis of K562 cells. Expression of FOXO3a, a novel C/EBPa-regulated gene, was required for apoptosis but not for differentiation induction or proliferation inhibition of K562 cells.
The transcription factor C/EBPα is more potent than C/EBPβ in inducing granulocitic differentiation and inhibiting BCR/ABL-expressing cells. We took a "domain swapping" approach to assess biological ...effects, modulation of gene expression, and binding to C/EBPα-regulated promoters by wild-type and chimeric C/EBPα/C/EBPβ proteins. Wild-type and N-C/EBPα+ C/EBPβ-DBD induced transcription of the granulocyte-colony stimulating factor receptor (G-CSFR) gene, promoted differentiation, and suppressed proliferation of K562 cells vigorously; instead, wild-type C/EBPβ and N-C/EBPβ+C/EBPα-DBD had modest effects, although they bound the G-CSFR promoter like wild-type C/EBPα and N-C/EBPα+C/EBPβ-DBD. Chimeric proteins consisting of the TAD of VP16 and the DBD of C/EBPα or C/EBPβ inhibited proliferation and induced differentiation of K562 cells as effectively as wild-type C/EBPα. Gene expression profiles induced by C/EBPα resembled those modulated by N-C/EBPα+C/EBPβ-DBD, whereas C/EBPβ induced a pattern similar to that of N-C/EBPβ+C/EBPα-DBD. C/EBPα activation induced changes in the expression of more cell cycle- and apoptosis-related genes than the other proteins and enhanced Imatinib-induced apoptosis of K562 cells. Expression of FOXO3a, a novel C/EBPα-regulated gene, was required for apoptosis but not for differentiation induction or proliferation inhibition of K562 cells.
Hematopoietic stem cells (HSCs) are responsible for the lifelong production and maintenance of adult blood cell types. They arise from the aorta at the embryonic day (E) 10.5 from a subset of ...endothelial cells that undergo to endothelial-to-hematopoietic transition (EHT). Our lab discovered that a G-coupled receptor protein, Gpr56, is required for the generation of HSCs in zebrafish. However, its role in mammalian hematopoiesis remains controversial. We investigate the functional role of Gpr56 in vitro using our mouse Gata2Venus (G2V) embryonic stem cell (ESC) line which allows us to highly enrich for hematopoietic progenitor cells (HPCs). G2V.G56-/-. We tested the Gpr56 loss of function by generating a full knock out (KO) Gpr56 mouse G2V (G2V.G56-/-) ESC line. The in vitro examination of phenotypic hematopoietic cell production by FACS and colony forming unit-culture (CFU-C) assay reported an increase in hematopoietic cells (HC) and HPCs production from G2V.G56-/- ESC as compared to G2V ESC. Further analyses performed on cell-sorted HPC population showed that another GPR protein, Gpr97 (located in the same locus), is highly expressed when Gpr56 is deleted. This led us to generate a novel G2V Gpr56/Gpr97 double knockout (G2V.G56/97-/-) ESC line. The in vitro differentiation of the G2V.G56/97-/- revealed a significant decrease in HPCs, supporting the idea that a compensatory mechanism by Gpr97 is taking place when Gpr56 is absent. Preliminary in vivo data using a Gpr56 conditional KO mouse model corroborates our hypothesis. The data demonstrate that the Gpr97 performs a redundant function upon deletion of Gpr56 in the HS/PC population and the deletion of both Gpr56 and Gpr97 is sufficient to impair the production of HS/PC and HC in vitro. We conclude that GPR signalling through Gpr56 and/or Gpr97 is required for HPC production.
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